The quantitative determination of the morphine content of opium by paper chromatography and paper electrophoresis

Abstract

Many different methods have been used to discover a very accurate micromethod for the quantitative determination of the morphine content of opium, and in our earlier work we tried using paper chromatography (Svendsen 1951, Svendsen Aarnes & Paulsen 1955). For a very accurate micromethod the following conditions must be fulfilled:

Many different methods have been used to discover a very accurate micromethod for the quantitative determination of the morphine content of opium, and in our earlier work we tried using paper chromatography (Svendsen 1951, Svendsen Aarnes & Paulsen 1955). For a very accurate micromethod the following conditions must be fulfilled:

The morphine must be completely extracted from the opium;

The morphine must be completely separated from other substances likely to affect the quantitative determination, and

A colour reaction or the like so far as possible specifically to morphine must be available for the quantitative determination of morphine

Since no colour reaction has apparently yet been evolved which is sufficiently specific for morphine for the effect of other substances to be disregarded, a very accurate determination is impossible unless the morphine is separated out as completely as possible. In paper chromatography morphine can now be satisfactorily separated from the other alkaloids present in appreciable quantities in opium by using suitable liquid mixtures, but it is difficult to say whether the morphine has been fully isolated from all substances likely to affect determination, since the quantities involved may be very small. However, the effect on the results of the analysis may also be fairly small.

No morphine is lost in quantitative paper chromatography and so the results given thereby, if all the morphine in the drug is extracted, may be higher but cannot be lower than the actual morphine content of the drug.

Recently paper electrophoresis has often been used successfully in separating alkaloid mixtures. Graf & List (1952) studied the separation of the alkaloids in opium by paper electrophoresis and found that under suitable conditions morphine will separate independently from the other alkaloids present in appreciable quantities in opium. However, it is not known whether the other substances present in the opium sample and likely to affect the quantitative determination of morphine in opium can be similarly separated.

We found it interesting to carry out comparative experiments on the quantitative determination of morphine in opium by paper chromatography and paper electrophoresis, our aim being to find out which of the two systems provided a better separation of morphine from the other alkaloids present in opium and also from other substances likely to affect the quantitative determination of morphine.

The main aim of our experiments was to effect a total extraction of the morphine in the opium. However, we did not carry out any comprehensive purifications of the opium extract produced before applying the chromatographic or electrophoretic test because we wanted to use whichever method proved the better in controlled experiments based on some of the official and unofficial methods used up to now to determine the morphine in opium, our aim being to find out exactly where the known methods broke down, and in experiments of the kind specified it is impossible to effect extensive purification of the opium extracts to be extracted (extraction residues, mother liquors from bialkaloid and morphine precipitates).

I. PAPER CHROMATOGRAPHY

Single-dimensional chromatography with a descending mobile phase was used. The atmosphere in the chromatography vessel was saturated by means of bowls placed on the base of the chamber and by means of "saturating paper" (Svendsen 1952). The chromatography paper used was Whatman filter paper no. 1 and the liquid system consisted of butanol, acetic acid, water, (4 : 1 : 5 parts by volume). When applied as a base or acetate, the morphine separates in this liquid system in the form of a smooth, clearly defined spot having an R f? value of 0.45. None of the known alkaloids present in appreciable quantities in opium and likely to react with the reagent (nitrite reagent) used for quantitative determinations takes up a position very near the morphine on the chromatogram.

Quantitative paper chromatography

Pure morphine base

Various quantities ( = 50 - 250 ?g of anhydrous morphine base) were sampled from a standard solution of pure morphine base in 5% acetic acid by means of a micropipette (Agla Micrometer Syringe, Burroughs Wellcome & Co., London) and applied to the chromatography paper. Not more than 10 ?1 were used per application. If larger quantities were required, a further 10 ?l were applied to the same spot after the first 10 ?1 applied had dried. The application could be repeated a number of times in the same way if required. The spots were applied about 3 cm apart approximately 10 cm from one narrow end of the chromatogram. The saturation time was 15-16 hours and the chromatogram formation time in the mobile liquid phase was 8-10 hours, the, temperature remaining at 20°C.

The stripe produced by the passage of one of the applied morphine spots was cut out of the chromatogram and developed with the nitrite reagent. Before the chromatogram was completely free of the acetic acid liquid mixture, it was sprayed with ? 5% aqueous solution of NaNO 2, the morphine spots appearing as yellowish nitrosomorphine. The colour changed to reddish-yellow when the chromatogram was suspended in an ammonia atmosphere. The place of the remaining morphine spots on the chromatogram could now be found easily and the paper containing the morphine cut out. The cut-out pieces of paper were always of the same size, since wide variations in size may affect the results.

The pieces of paper were cut up into smaller pieces and placed in a test tube. 2 ml of 1% hydrochloric acid and 2 ml of 0.5% NaNO 2were added by means of a pipette, 1 ml of 5% ammonia solution being added 15 minutes later. The resultant colour solution was transferred to a basin of 10 mm diameter and the extinction was measured in a Lumetron colorimeter using an M 465 filter. The correction curve for anhydrous morphine base was linear.

Morphine in opium

Preparation of the opium extract for quantitative determination : 0.5 g of pulverized opium (recently weighed) was carefully ground in a small mortar or porcelain vessel with 1 ml of water until a homogeneous mixture was formed, and then, in the same way, with 10 g of acetous aluminium oxide (Woelm, Eschwege). The dry mixture of opium and aluminium oxide was transferred quantitatively to a 12 mm diameter chromatography tube and extracted with water until a total of 25.0 ml of filtrate was collected (measuring flask).

Extracts were made of the slightly yellow opium extract prepared and different quantities were applied in the same way as just described for the quantitative determination. The stripes produced by the passage of the applied and chromatographed opium extracts showed up when the chromatograms were examined in ultra-violet light, since the opium extract contains a number of substances which fluoresce in ultra-violet light. One of the stripes was developed with the nitrite reagent, while the other morphine spots were cut out of the chromatogram and the quantitative determination of morphine performed as previously described. We found that the work of finding the morphine stripe when the latter was applied alone was made easier by applying a small quantity of papaverin together with the morphine prior to the chromatographic separation. Papaverin passes through much more slowy than morphine and fluoresces a distinctive blue colour under ultra-violet light.

The results of the determinations of 4 opium samples tested using the paper chromatography method are given in table 1. All the figures in the table are percentage morphine contents and are the averages of 8 analyses.

In the case of opium 4 it proved necessary to weigh only 0.2 - 0.3 g of opium to obtain a total extraction of the morphine. The values with 0.5 g of opium were too low.

TABLE 1

Opium 1

Opium 2

Opium 3

Opium 4

12.2

12.2

12.5

19.2

12.2

12.6

12.3

19.2

12.4

12.5

12.6

12.4

12.6

12.3

By way of comparison, table 2 gives the results of morphine determinations on the same four opium samples in accordance with Mannich's method ( Pharmacopoea suecica 1946), except that chloro-dinitrobenzene was replaced by fluoro-dinitro-benzene which reacts more quickly.

TABLE 2

Opium 1

Opium 2

Opium 3

Opium 4

12.7

12.8

12.8

18.4

12.4

12.8

12.8

18.5

It will be apparent from tables 1 and 2 that the values given by paper chromatography are lower than those given by Mannich's method, except in the case of opium 4 where the morphine extraction using Mannich's method is incomplete.

II. PAPER ELECTROPHORESIS

A paper electrophoresis apparatus of the "Electrorheophor" type (Gerard Pleuger, Antwerp) and 18 ? 40 cm pieces of Whatman filter paper No. 1 were used. 10% acetic acid was used as buffer liquid. The morphine - that is, the opium extract - was applied as previously described to dry paper about 10 cm from its narrow end, which should be placed in the anode chamber. The paper was then placed in the apparatus and sprayed as uniformly as possible with the buffer liquid used in the electrophoresis. After the paper had been moistened uniformly, a 500 V current was applied and electrophoresis continued for three hours. The pherogram was then taken out and the rest of the process was the same as for the paper chromatography experiments.

The morphine separated in the form of a smooth clearly defined spot and was undisturbed by the other main alkaloids in the opium. The correction curve for the pure anhydrous morphine base was linear and coincided with the adjusting curve for the paper chromatographed anhydrous morphine base.

The results of the paper electrophoresis method for the same opium samples as were previously used are given in table 3, where the figures are again an average derived from a series of separate analysis (6).

TABLE 3

Opium 1

Opium 2

Opium 3

Opium 4

13.3

13.1

13.4

20.7

13.7

13.4

13.4

20.5

13.7

13.3

13.2

13.5

13.4

Conclusions

The results given by paper chromatography and paper electrophoresis are of a satisfactory accuracy, although the electrophoresis results are very regularly appreciably higher than those given by paper chromatography. In the light of what is stated in the introduction, the inference apparently is that separation by paper chromatography is better than the paper electrophoresis method in the conditions used. The use of different buffer solutions and pH factors in the electro- phoretic separation process did not lead to better separation and lower morphine values.